Molecular Basis for E-cadherin Recognition by Killer Cell Lectin-like Receptor G1 (KLRG1)

Seiko Nakamura,Kimiko Kuroki,Izuru Ohki,Kaori Sasaki,Mizuho Kajikawa,Takuma Maruyama,Masayuki Ito,Yosuke Kameda,Mitsuhiko Ikura,Kazuo Yamamoto,Naoki Matsumoto,Katsumi Maenaka

doi:10.1074/jbc.m109.038802

Abstract

The killer cell lectin-like receptor G1, KLRG1, is a cell surface receptor expressed on subsets of natural killer (NK) cells and T cells. KLRG1 was recently found to recognize E-cadherin and thus inhibit immune responses by regulating the effector function and the developmental processes of NK and T cells. E-cadherin is expressed on epithelial cells and exhibits Ca(2+)-dependent homophilic interactions that contribute to cell-cell junctions. However, the mechanism underlying the molecular recognition of KLRG1 by E-cadherin remains unclear. Here, we report structural, binding, and functional analyses of this interaction using multiple methods. Surface plasmon resonance demonstrated that KLRG1 binds the E-cadherin N-terminal domains 1 and 2 with low affinity (K(d) approximately 7-12 microm), typical of cell-cell recognition receptors. NMR binding studies showed that only a limited N-terminal region of E-cadherin, comprising the homodimer interface, exhibited spectrum perturbation upon KLRG1 complex formation. It was confirmed by binding studies using a series of E-cadherin mutants. Furthermore, killing assays using KLRG1(+)NK cells and reporter cell assays demonstrated the functional significance of the N-terminal region of E-cadherin. These results suggest that KLRG1 recognizes the N-terminal homodimeric interface of domain 1 of E-cadherin and binds only the monomeric form of E-cadherin to inhibit the immune response. This raises the possibility that KLRG1 detects monomeric E-cadherin at exposed cell surfaces to control the activation threshold of NK and T cells.

Highlights

Natural killer (NK)3 cells play a critical role in the innate immune system because of their ability to kill other cells
Preparation of Recombinant Proteins—N-terminal domains 1 and 2 of M. musculus E-cadherin with a His10 tag, a spacer sequence, and a Factor Xa recognition site at the N terminus were expressed in E. coli either as a soluble protein or inclusion bodies
This study shows that KLRG1 recognizes a relatively broad area of the N-terminal region of E-cadherin (Trp2–Pro6), which largely overlaps with the strand-exchanged homodimer interface

Summary

Introduction

Natural killer (NK) cells play a critical role in the innate immune system because of their ability to kill other cells. The integration of the signals from these receptors determines the final functional outcome of NK cells These inhibitory and activating receptors can be divided into two structurally different groups, the Ig-like receptors and the C-type lectin-like receptors, based on the structural aspects of their extracellular regions. The Ig-like receptors include killer cell Ig-like receptors and the leukocyte Ig-like receptors, and the C-type lectin-like receptors include CD94/ NKG2(KLRD/KLRC), Ly49(KLRA), NKG2D(KLRK), NKRP1(KLRB), and KLRG1 Many of these immune receptors recognize major histocompatibility complex class I molecules or their relatives [2,3,4], but there are still many orphan receptors expressed on NK cells. E-cadherin is primarily expressed on epithelial cells, and its extracellular region consists of several domains that include cadherin motifs [15, 16] These domains mediate Ca2ϩ-dependent homophilic interactions to facilitate cell adhesion. The N-terminal region plays important roles in homophilic binding and cell adhesion

Results

Discussion

Conclusion